Ca
2+
-influx through L-type Ca
2+
-channels (LTCCs) is associated with activity-related stressful oscillations of Ca
2+
levels within dopaminergic (DA) neurons in the substantia nigra (SN), which ...may contribute to their selective degeneration in Parkinson's disease (PD). LTCC blockers were neuroprotective in mouse neurotoxin models of PD, and isradipine is currently undergoing testing in a phase III clinical trial in early PD. We report no evidence for neuroprotection by
in vivo
pretreatment with therapeutically relevant isradipine plasma levels, or Ca
v
1.3 LTCC deficiency in 6-OHDA-treated male mice. To explain this finding, we investigated the pharmacological properties of human LTCCs during SN DA-like and arterial smooth muscle (aSM)-like activity patterns using whole-cell patch-clamp recordings in HEK293 cells (Ca
v
1.2 α1-subunit, long and short Ca
v
1.3 α1-subunit splice variants; β3/α2δ1). During SN DA-like pacemaking, only Ca
v
1.3 variants conducted Ca
2+
current (I
Ca
) at subthreshold potentials between action potentials. SN DA-like burst activity increased integrated I
Ca
during (Ca
v
1.2 plus Ca
v
1.3) and after (Ca
v
1.3) the burst. Isradipine inhibition was splice variant and isoform dependent, with a 5- to 11-fold lower sensitivity to Ca
v
1.3 variants during SN DA-like pacemaking compared with Ca
v
1.2 during aSM-like activity. Supratherapeutic isradipine concentrations reduced the pacemaker precision of adult mouse SN DA neurons but did not affect their somatic Ca
2+
oscillations. Our data predict that Ca
v
1.2 and Ca
v
1.3 splice variants contribute differentially to Ca
2+
load in SN DA neurons, with prominent Ca
v
1.3-mediated I
Ca
between action potentials and after bursts. The failure of therapeutically relevant isradipine levels to protect SN DA neurons can be explained by weaker state-dependent inhibition of SN DA LTCCs compared with aSM Ca
v
1.2.
SIGNIFICANCE STATEMENT
The high vulnerability of dopamine (DA) neurons in the substantia nigra (SN) to neurodegenerative stressors causes Parkinson's disease (PD). Ca
2+
influx through voltage-gated L-type Ca
2+
channels (LTCCs), in particular Ca
v
1.3, appears to contribute to this vulnerability, and the LTCC inhibitor isradipine is currently being tested as a neuroprotective agent for PD in a phase III clinical trial. However, in our study isradipine plasma concentrations approved for therapy were not neuroprotective in a PD mouse model. We provide an explanation for this observation by demonstrating that during SN DA-like neuronal activity LTCCs are less sensitive to isradipine than Ca
v
1.2 LTCCs in resistance blood vessels (mediating dose-limiting vasodilating effects) and even at supratherapeutic concentrations isradipine fails to reduce somatic Ca
2+
oscillations of SN DA neurons.
Androgen receptor targeted therapies have emerged as an effective tool to manage advanced prostate cancer (PCa). Nevertheless, frequent occurrence of therapy resistance represents a major challenge ...in the clinical management of patients, also because the molecular mechanisms behind therapy resistance are not yet fully understood. In the present study, we therefore aimed to identify novel targets to intervene with therapy resistance using gene expression analysis of PCa co-culture spheroids where PCa cells are grown in the presence of cancer-associated fibroblasts (CAFs) and which have been previously shown to be a reliable model for antiandrogen resistance.
Gene expression changes of co-culture spheroids (LNCaP and DuCaP seeded together with CAFs) were identified by Illumina microarray profiling. Real-time PCR, Western blotting, immunohistochemistry and cell viability assays in 2D and 3D culture were performed to validate the expression of selected targets in vitro and in vivo. Cytokine profiling was conducted to analyze CAF-conditioned medium.
Gene expression analysis of co-culture spheroids revealed that CAFs induced a significant upregulation of cholesterol and steroid biosynthesis pathways in PCa cells. Cytokine profiling revealed high amounts of pro-inflammatory, pro-migratory and pro-angiogenic factors in the CAF supernatant. In particular, two genes, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 (HMGCS2) and aldo-keto reductase family 1 member C3 (AKR1C3), were significantly upregulated in PCa cells upon co-culture with CAFs. Both enzymes were also significantly increased in human PCa compared to benign tissue with AKR1C3 expression even being associated with Gleason score and metastatic status. Inhibiting HMGCS2 and AKR1C3 resulted in significant growth retardation of co-culture spheroids as well as of various castration and enzalutamide resistant cell lines in 2D and 3D culture, underscoring their putative role in PCa. Importantly, dual targeting of cholesterol and steroid biosynthesis with simvastatin, a commonly prescribed cholesterol synthesis inhibitor, and an inhibitor against AKR1C3 had the strongest growth inhibitory effect.
From our results we conclude that CAFs induce an upregulation of cholesterol and steroid biosynthesis in PCa cells, driving them into AR targeted therapy resistance. Blocking both pathways with simvastatin and an AKR1C3 inhibitor may therefore be a promising approach to overcome resistances to AR targeted therapies in PCa. Video abstract.
Our basic understanding of ascending thoracic aortic aneurysm (ATAA) pathogenesis is still very limited, hampering early diagnosis, risk prediction, and development of treatment options. ..."Omics"-technologies, ideal to reveal tissue alterations from the normal physiological state due to disease have hardly been applied in the field. Using a metabolomic approach, with this study the authors seek to define tissue differences between controls and various forms of ATAAs.
Using a targeted FIA-MS/MS metabolomics approach, we analysed and compared the metabolic profiles of ascending thoracic aortic wall tissue of age-matched controls (n = 8), bicuspid aortic valve-associated aneurysms (BAV-A; n = 9), tricuspid aortic valve-associated aneurysms (TAV-A; n = 14), and tricuspid aortic valve-associated aortic dissections (TAV-Diss; n = 6).
With sphingomyelin (SM) (OH) C22:2, SM C18:1, SM C22:1, and SM C24:1 only 4 out of 92 detectable metabolites differed significantly between controls and BAV-A samples. Between controls and TAV-Diss samples only phosphatidylcholine (PC) ae C32:1 differed. Importantly, our analyses revealed a general increase in the amount of total sphingomyelin levels in BAV-A and TAV-Diss samples compared to controls.
Significantly increased levels of sphingomyelins in BAV-A and TAV-Diss samples compared to controls may argue for a repression of sphingomyelinase activity and the sphingomyelinase-ceramide pathway, which may result in an inhibition of tissue regeneration; a potential basis for disease initiation and progression.
Proton-transfer reaction mass spectrometry (PTR-MS) is a versatile tool for the mass spectrometric analysis of organic molecules in gaseous samples. Due to its operation principle, PTR-MS is a soft ...ionization technique generating spectral data typically rich in protonated molecule information. Most of the currently reported PTR-MS applications are designed to determine volatile compounds. Herein, we present a redesigned instrumental setup termed “high-temperature (HT)-PTR-MS” with improved capabilities for the analysis of low-volatile compounds. The developed HT-PTR-MS prototype was successfully hyphenated with gas chromatography (GC) to enable qualitative and quantitative analysis of licit and illicit drugs in human blood/plasma samples. Different kinds of spiked and authentic samples were used to evaluate the performance of the GC-HT-PTR-MS in forensic drug testing. Benchmarking against GC-MS with electron ionization demonstrated the improved detection capabilities of GC-HT-PTR-MS in screening applications. On average, one order of magnitude lower limits of detection/identification were reached. Clearly, GC-HT-PTR-MS has the vast potential to complement or even replace established mass spectrometric techniques in forensic drug analysis.
An integrated platform to screen crude extracts of the red alga Laurencia aiming at the dereplication of known metabolites and the accelerated detection of new natural products. Display omitted
•An ...integrated approach for the dereplication of Laurencia extracts was developed.•A searchable ‘in-house’ Laurencia-focused NMR database was constructed.•An ‘in-house’ Laurencia-focused LC–MS library is being built.•Two new C15 bromoallene acetogenins were isolated from Laurencia chondrioides.
The global metabolic profile of Laurencia crude red algal extracts was addressed by applying high-throughput analytical techniques, namely UHPLC–PDA–HRMS and 2D HSQC NMR. An integrated platform including software tools and databases, such as Xcalibur, ToxID, ACD/Labs and MarinLit, has been developed to mine the complex analytical data towards the accelerated identification of known metabolites and the detection of new natural products at the early stages of phytochemical analysis. In parallel, a searchable ‘in-house’ Laurencia-focused NMR database incorporating chemical structures, NMR spectroscopic data and reported biological activities has been generated. The screening strategy has been developed as a tool to prioritize the crude extracts to be further subjected to phytochemical analysis by tracing the presence of new natural products among the pool of known compounds. The successful application of this integrated methodology in the crude extract of Laurencia chondrioides led to the rapid detection of two new C15 bromoallene acetogenins (1 and 2), which were subsequently isolated and characterized.
Germline de novo missense variants of the CACNA1D gene, encoding the pore-forming α1 subunit of Cav1.3 L-type Ca2+ channels (LTCCs), have been found in patients with neurodevelopmental and endocrine ...dysfunction, but their disease-causing potential is unproven. These variants alter channel gating, enabling enhanced Cav1.3 activity, suggesting Cav1.3 inhibition as a potential therapeutic option. Here we provide proof of the disease-causing nature of such gating-modifying CACNA1D variants using mice (Cav1.3AG) containing the A749G variant reported de novo in a patient with autism spectrum disorder (ASD) and intellectual impairment. In heterozygous mutants, native LTCC currents in adrenal chromaffin cells exhibited gating changes as predicted from heterologous expression. The A749G mutation induced aberrant excitability of dorsomedial striatum-projecting substantia nigra dopamine neurons and medium spiny neurons in the dorsal striatum. The phenotype observed in heterozygous mutants reproduced many of the abnormalities described within the human disease spectrum, including developmental delay, social deficit, and pronounced hyperactivity without major changes in gross neuroanatomy. Despite an approximately 7-fold higher sensitivity of A749G-containing channels to the LTCC inhibitor isradipine, oral pretreatment over 2 days did not rescue the hyperlocomotion. Cav1.3AG mice confirm the pathogenicity of the A749G variant and point toward a pathogenetic role of altered signaling in the dopamine midbrain system.
The small alpine district of East Tyrol (Austria) has an exceptional demographic history. It was contemporaneously inhabited by members of the Romance, the Slavic and the Germanic language groups for ...centuries. Since the Late Middle Ages, however, the population of the principally agrarian-oriented area is solely Germanic speaking. Historic facts about East Tyrol's colonization are rare, but spatial density-distribution analysis based on the etymology of place-names has facilitated accurate spatial mapping of the various language groups' former settlement regions. To test for present-day Y chromosome population substructure, molecular genetic data were compared to the information attained by the linguistic analysis of pasture names. The linguistic data were used for subdividing East Tyrol into two regions of former Romance (A) and Slavic (B) settlement. Samples from 270 East Tyrolean men were genotyped for 17 Y-chromosomal microsatellites (Y-STRs) and 27 single nucleotide polymorphisms (Y-SNPs). Analysis of the probands' surnames revealed no evidence for spatial genetic structuring. Also, spatial autocorrelation analysis did not indicate significant correlation between genetic (Y-STR haplotypes) and geographic distance. Haplogroup R-M17 chromosomes, however, were absent in region A, but constituted one of the most frequent haplogroups in region B. The R-M343 (R1b) clade showed a marked and complementary frequency distribution pattern in these two regions. To further test East Tyrol's modern Y-chromosomal landscape for geographic patterning attributable to the early history of settlement in this alpine area, principal coordinates analysis was performed. The Y-STR haplotypes from region A clearly clustered with those of Romance reference populations and the samples from region B matched best with Germanic speaking reference populations. The combined use of onomastic and molecular genetic data revealed and mapped the marked structuring of the distribution of Y chromosomes in an alpine region that has been culturally homogeneous for centuries.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an indispensable analytical technique in clinical and forensic toxicology for detection and identification of potentially toxic or ...harmful compounds. Particularly, non-target LC-MS/MS assays enable extensive and universal screening requested in systematic toxicological analysis. An integral part of the identification process is the generation of information-rich product ion spectra which can be searched against libraries of reference mass spectra. Usually, ‘data-dependent acquisition’ (DDA) strategies are applied for automated data acquisition. In this study, the ‘data-independent acquisition’ (DIA) method ‘Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra’ (SWATH) was combined with LC-MS/MS on a quadrupole-quadrupole-time-of-flight (QqTOF) instrument for acquiring informative high-resolution tandem mass spectra. SWATH performs data-independent fragmentation of all precursor ions entering the mass spectrometer in 21
m/z
isolation windows. The whole
m/z
range of interest is covered by continuous stepping of the isolation window. This allows numerous repeat analyses of each window during the elution of a single chromatographic peak and results in a complete fragment ion map of the sample. Compounds and samples typically encountered in forensic casework were used to assess performance characteristics of LC-MS/MS with SWATH. Our experiments clearly revealed that SWATH is a sensitive and specific identification technique. SWATH is capable of identifying more compounds at lower concentration levels than DDA does. The dynamic range of SWATH was estimated to be three orders of magnitude. Furthermore, the >600,000 SWATH spectra matched led to only 408 incorrect calls (false positive rate = 0.06 %). Deconvolution of generated ion maps was found to be essential for unravelling the full identification power of LC-MS/MS with SWATH. With the available software, however, only semi-automated deconvolution was enabled, which rendered data interpretation a laborious and time-consuming process.
Graphical Abstract
High-resolution LC-MS/MS with SWATH represents a sensitive and specific compound identification tool that has vast potential to become a leading technique in systematic toxicological analysis. SWATH solves the problem of unused precursor ions often encountered with data-dependent acquisition methods by acquiring complete fragment ion maps of a sample
Hexahydrocannabinol (HHC), 6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydrobenzocchromen-1-ol, is a semi-synthetic cannabinoid that has presented challenges to analytical laboratories due to its ...emergence and spread in the drug market. The lack of information on human pharmacokinetics hinders the development and application of presumptive and confirmatory tests for reliably detecting HHC consumption. To address this knowledge gap, we report the analytical results obtained from systematic forensic toxicological analysis of body-fluid samples collected from three individuals suspected of drug-impaired driving after HHC consumption. Urine and plasma samples were analyzed using non-targeted liquid chromatography-high-resolution tandem mass spectrometry. The results provided evidence that HHC undergoes biotransformation reactions similar to other well-characterized cannabinoids, such as ∆9-tetrahydrocannabinol or cannabidiol. Notably, HHC itself was only detectable in plasma samples, not in urine samples. The observed Phase I reactions involved oxidation of C11 and the pentyl side chain, leading to corresponding hydroxylated and carboxylic acid species. Additionally, extensive glucuronidation of HHC and its Phase I metabolites was evident.
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